台灣金線連免疫調節蛋白之純化與活性分析

Purification and Function Analysis of the Immunomodulatory Protein from Anoectochilus formosanus

張文瀠(Wen-Ying Chang)

指導教授 ： 許輔

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.02491

摘要

台灣金線連 (Anoectochilus formosanus Hayata) 經硫酸銨沈澱以及DE-52 / Hi-Trap Q離子交換層析，可以純化出一個66 kDa及一個14 kDa之蛋白以及一種未知紅色化合物，稱為台灣金線連免疫調節區分物（immunomodulatory fraction from A. formosanus Hayata, IFAF）。IFAF並非醣蛋白，亦不具有凝集小鼠紅血球之活性。IFAF可活化巨噬細胞，促進該細胞分泌NO及TNF-alpha，並提高iNOS, TNF-alpha, IL-1 beta

關鍵字

免疫調節 ； 台灣金線連

並列摘要

IFAF（immunomodulatory fraction from Anoectochilus formosanus Hayata） was purified by ammonium sulfate precipitation and DE-52 / HiTrap Q ion-exchange chromatography from A. formosanus. IFAF includes two proteins with molecular weight of 66 kDa and 14 kDa and an unclear red compound. Neither is IFAF a glycoprotein nor does it agglutinate mouse red blood cells. IFAF activates RAW 264.7 macrophages by enhancing the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha), and also increases the expression of mRNAs of iNOS, TNF-alpha, IL-1 beta, and IL-18 by the cells. Additionally, IFAF also activates murine splenocytes. It stimulates the proliferation of murine splenocytes, increases the gamma-interferon (IFN-gamma) secretion, and promotes the mRNA expression of IL-2, IL-5, and IFN-gamma. Moreover three hybridoma clones that secrete monoclonal antibodies recognizing different protein components of IFAF are obtained. In order to understand the component of IFAF, the proteins are precipitated by acetone to exclude the red compounds, and the proteins with molecular weight of 66 kDa and 14 kDa are found in the acetone-precipitant. These proteins are further separated using isoelectric focusing techniques. The active component of IFAF is determinated by autoclave, trypsin hydrolysis and antibody neutralization. The results support that the 14 kDa protein has immunomodulatory ability. Therefore, the 14 kDa protein is further named as IPAF (immunomodulatory protein from A. formosanus Hayata). IPAF is an important biofunctional component of A. fomosanus, with medicinal capability and could strongthen the immunity of the host.

並列關鍵字

Anoectochilus formosanus ； immunomodulatory

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